BATTERIOLOGICA DIAGNOSI

DIAGNOSI

BATTERIOLOGICA

Raccolta e trasporto del campione

L’attendibilità dell’esame batteriologico dipende da vari fattori:

• ADEGUATA RACCOLTA DEL CAMPIONE

• TRASPORTO DEL CAMPIONE:

terreno semisolido di Stuart o di Amies; Amies

con carbone vegetale;contenitori adatti per

anaerobi

DIAGNOSI COLTURALE

L’identificazione delle colonie viene eseguita ricorrendo a colture pure derivate da ciascun tipo di colonia precedentemente isolata e si basa sullo studio di alcuni caratteri:

 CARATTERI CULTURALI: ASPETTO COLONIE

 CARATTERI MICROSCOPICI: (morfologia e caratteristiche tintoriali)

 CARATTERI BIOCHIMICI: (fermentazione zuccheri;

produzione di prodotti metabolici peculiari;

presenza/assenza di enzimi particolari)

 CARATTERI ANTIGENICI

In base alle proprietà selettive o differenziali

i terreni vengono distinti in:

1.non selettivi 2.elettivi

3.selettivi

4.differenziali

Terreni non selettivi

I substrati non selettivi

consentono

(almeno in teoria) di coltivare tutti i microrganismi. Sono in genere

substrati ricchi, a pH neutro. Esempi:

–

cavallo o montone)

–

particolarmente esigenti

–

(Standard) un substrato per la determinazione del tenore in germi in prodotti alimentari

–

substrato ricco per la coltivazione

dei batteri con elevate esigenze

nutrizionali

Terreni elettivi

I substrati elettivi hanno una composizione che favorisce, anche se non in modo assoluto, un determinato gruppo di microrganismi.

Sono terreni ricchi di nutrienti, spesso con la presenza di sangue di pecora o cavallo, che consentono la crescita di quasi tutte le specie batteriche di interesse medico.

Esempi:

a.

un substrato elettivo per gli streptococchi lattici,

ricco in nutrienti e tamponato

TERRENI SELETTIVI

Consentono la crescita selettiva di determinati microrganismi; vengono utilizzati quando si trattano campioni microbiologici contenenti popolazioni batteriche differenti.

Per poter rendere selettivo un terreno si può operare su vari parametri:

A.CONDIZIONI NUTRIZIONALI B.PH

C.TEMPERATURA

D.INIBITORI

TERRENI SELETTIVI E DIFFERENZIALI

Selettivi perché favoriscono la crescita di determinate specie batteriche inibendone altre

Differenziali perché permettono la crescita di

tipi diversi di microrganismi che però crescendo

formano colonie facilmente distinguibili (per

esempio in base alla colorazione)

Luria Bertani Broth (LB)

LB-Agar Sheep's

Blood Agar Chocolate

Agar MacConkey

Agar Hektoen

Enteric Agar

Common Microbiological Media for Aerobic Cultures

Uses:

Culture of aerobic bacterial species

Uses: Culture of aerobic bacterial

species on solid media

Uses: Non-

selective media, good for

culturing many bacterial

species including Gram negative and Gram-

positive species including

Staphylococci and Streptococci

Uses: Very enriched media with lysed red cells, to release many nutrients needed by

*fastidious*

organisms.

Uses: Selective media for Gram- negative,

enteric bacteria (bile tolerant).

Assess lactose fermentation

Uses: Selective media for Gram- negative enteric bacteria (higher bile salt concentrations than MacConkey).

Used to assess lactose

fermentation and ability to produce hydrogen sulfide (H2S). Allows for distinguishing among, E. coli, Salmonella and other gut pathogens including Shigella species

This enriched and differential plating medium supports the growth of most medically significant bacteria. It is used for primary plating and for subculturing of colonies and is especially useful for detecting hemolytic activity of bacteria. It is also used

for the demonstration of the "satellite phenomenon" by

Haemophilus

and for the CAMP test on b-hemolytic streptococci.

Blood Agar Sheep

Blood Agar: Hemolytic Reactions

When streaked on Blood Agar, many species of bacteria cause hemolysis – i.e., destruction of the erythrocytes (and hemoglobin) in the medium. Hemolytic reactions are generally classified as alpha, beta or gamma according to the appearance of zones around isolated colonies growing on or in the medium:

• Beta hemolysis: The colony is surrounded by a white or clear zone in which few or no intact erythrocytes are found. This reaction is best seen when the organism is growing under anaerobic conditions. Beta hemolysis is caused by one or more erythrocyte-lysing enzymes called hemolysins.

• Alpha hemolysis: The colony is surrounded by a zone of intact but discolored erythrocytes that have a greenish color. This appearance is generally due to the action of peroxide produced by the bacteria.

• Gamma hemolysis is simply a synonym for negative hemolysis

in which there is no change in the medium surrounding the

colony

Blood Agar: Hemolytic Reactions

• Beta hemolysis : In the top photo at right which shows

growing on Blood Agar, the light to clear zones can be seen around the colonies. Where growth occurs in the presence of less oxygen the reaction is more pronounced.

• Alpha hemolysis: ^{The bottom}

photo shows

growing on Blood Agar; some greenish

zones of alpha hemolysis are visible.

Blood Agar Sheep

Formazione di zone di emolisi intorno a colonie che producono emolisine su agar sangue.

A sinistra Streptococcus mitis, un batterio a-emolitico

A destra Streptococcus pyogenes di gruppo A (GAS), tipico b-emolitico

Blood Agar Sheep

Blood Agar

Colonies of Pseudomonas aeruginosa typically display beta hemolysis,

a metallic sheen, and blue or green pigment.

Blood Agar

(with 5% Sheep Blood)

OPTOCHINA BACITRACINA

CAMP test on b-hemolytic streptococci (gruppo B)

S. agalactiae strains produce a diffusible, extracellular compound (CAMP factor) that will, in conjunction with a specific beta-hemolysin of Staphylococcus aureus,

cause complete lysis of sheep red blood cells in an agar medium.

In this photo, a Blood Agar plate is shown after 24 hours of incubation at 37°C.

The vertical streak is a beta-hemolysin-producing strain of Staphylococcus aureus, and at right angles to it are streaks of (1)Enterococcus faecalis, (2)Streptococcus salivarius, (3)S.

agalactiae, and (4) E. durans.

Note the large area of complete lysis where the extracellular compound of S. agalactiae encounters the beta-lysin of S. aureus.

Streptococcus agalactiae

Staphylococcus aureus Enterococcus faecalis

Streptococcus salivarius Enterococcus durans

CAMP test on b-hemolytic streptococci

(gruppo B)

Blood Agar Sheep

E’ conosciuta anche come Salmonella arizonae, batterio zoonosico che può infettare uomo, uccelli, rettili, ed altri animali.

Colonie di Salmonella choleraesuis subsp. arizonae

su a blood agar

CHOCOLATE AGAR

Chocolate agar is a nutrient medium which is used in culturing fastidious

organisms such as Haemophilus species and Neisseria. It is comprised

of sheep blood that provides the X and V factors necessary for

Medium proposed by THAYER and MARTIN (1964, 1966) for the selective isolation of Neisseria gonorrhoeae and Neisseria meningitidis from clinical specimens.

Addition of various antibiotics (THAYER-MARTIN Supplement I), and specific growth-promoting agents (THAYER-MARTIN Supplement II) to the basic substrate with or without hemoglobin usually inhiits or totally suppresses the growth of accompanying bacterial flora, while allowing pathogenic Neisseria species to grow normally.

THAYER-MARTIN Supplement I: vancomicina, colistina, nistatina e trimetoprim lattato

THAYER MARTIN AGAR

TRYPTIC SOY BROTH/AGAR

Tryptic soy agar and broth is a basic medium used for culturing many kinds of microorganisms. Tryptic soy agar is mainly used as an initial growth medium for the following purposes:

•observe colony morphology

•develop a pure culture

•achieve sufficient growth for further biochemical testing

•culture storage

It can also be used in the determination of bacterial numbers.

substrato ricco non selettivo per la coltivazione dei batteri con elevate esigenze nutrizionali

Pseudomonas aeruginosa

su agar soy trypticase

AGAR SALMONELLA-SHIGELLA

Contiene:

1.Sali biliari, verde brillante e sodio citrato che rendono il terreno selettivo poiché inibiscono la crescita di Gram+

2.Lattosio con indicatore di pH (rosso neutro) che rende il terreno differenziale poiché la Salmonella e la Shigella non fermentano il lattosio a differenza di altri Gram-.

Quindi su questo terreno potranno crescere tutti i Gram-;

ma la Salmonella e la Shigella daranno colonie incolori

mentre i batteri che fermentano il lattosio daranno

colonie di colore rosa.

AGAR SALMONELLA-SHIGELLA

Colonie lattosio negative sono incolori Colonie lattosio positive sono rosa

Colonie di microrganismi che producono H2S sono nere al centro

(Alcune specie dei generi Proteus e Salmonella presentano colonie con centro nero; ciò è dovuto alla precipitazione del ferro solfuro, indotta dalla produzione di idrogeno solforato a partire dal sodio tiosolfato presente nel terreno.)

•Salmonella typhimurium

AGAR SALMONELLA-SHIGELLA

AGAR MS (salato al mannitolo)

E’ selettivo perché presenta una elevata concentrazione di NaCl che inibisce la maggior parte dei batteri tranne quelli sale-tolleranti come gli Stafilococchi.

Il mannitolo rende il terreno differenziale: questo zucchero infatti viene fermentato dagli Stafilococchi patogeni che daranno origine a colonie giallastre

Utilizzato per isolare Stafilococchi patogeni. L’acido prodotto dalla fermentazione del mannitolo determina il viraggio dell’indicatore di pH (rosso fenolo) da rosso (alcalino) a giallo (acido). Gli stafilococchi non patogeni sono capaci di crescere sul mezzo ma non producono acido.

TOLLERANO ELEVATE CONCENTRAZIONI DI NaCl (germi ALOFILI);

LA MAGGIOR PARTE DEI CEPPI PATOGENI FERMENTA LA MANNITE AGAR SALE MANNITE: terreno utilizzato per l’isolamento di Stafilococchi patogeni:

CONTIENE 7.5% NaCl che inibisce la crescita della maggior parte degli altri microrganismi.

CONTIENE LA MANNITE COME UNICA FONTE DI CARBONIO

Stafilococchi su AGAR SALE MANNITE

S. aureus:

colonie acidificanti per fermentazione della

mannite e viraggio al giallo dell’indicatore rosso fenolo S. epidermidis:

colonie alcaline (rosa);

non fermentanti la mannite

MAcCONKEY AGAR

MacConkey agar is probably the most popular solid differential medium in the world.

It is mainly used in identification of lactose fermenting, Gram-negative enteric pathogens and for inhibiting growth of Gram-positive organisms. Bacterial colonies that can ferment lactose turn the medium red. This red color is due to the pH indicators response to the acidic environment created by fermenting lactose.

Organisms that do not ferment lactose do not cause a color change.

Sali biliari (1,5%) e cristal violetto:

inibiscono i Gram + Indicatore di PH:rosso neutro

LATTOSIO

a) Serratia marcescens su agar MacConkey

Shigella flexneri su agar MacConkey MAcCONKEY AGAR

HEKTOEN ENTERIC (HE) AGAR

Indicatore di PH

Colonie lattosio non fermentanti

S. typhimurium su HE agar forma colonie blu-verdi non

fermentanti il lattosio, inoltre produce idrogeno solforato (H₂S), come evidenziato dal deposito nero al centro delle colonie.

HE agar è un mezzo utilizzato per isolare i batteri fecali della famiglia delle Enterbacteriaceae.

Colonie di Salmonella typhimurium su Hektoen enteric (HE) agar

HEKTOEN ENTERIC (HE) AGAR

Hektoen enteric agar is a medium designed for the isolation and recovery of fecal specimens belonging to the Enterbacteriaceae family, especially Salmonella and Shigella. It can differentiate between bacteria that ferment lactose and those that don't. Acid produced from fermenting lactose imparts a yellow-orange color to the medium due to the presence of a pH indicator. Non-lactose fermenters do not significantly change the color of the medium. HE agar can also detect the production of hydrogen sulfide gas, which turns parts of the medium black.

HEKTOEN ENTERIC (HE) AGAR

HEKTOEN ENTERIC (HE) AGAR

Klebsiella pneumoniae ferments lactose and produces salmon- colored growth. Micrococcus luteus does not grow. The white specks are crystals formed by a component of the media that precipitated out of solution when the plates were cooled. They look somewhat like a contaminant, but they do not interfere with the performance of the media.

EOSIN METHYLENE BLUE (EMB) AGAR

Eosin methylene blue agar, as the name suggests, contains the dyes eosin and methylene blue. EMB agar is selective because the aniline dyes in this purple media inhibit growth of Gram-positive organisms. Lactose fermenters metabolize the lactose in the media and produce acid byproducts, causing a color change in the colony. Thus, EMB is also a differential medium.

Strong acid production by organisms such as E. coli results in a metallic green sheen.

Weaker fermentation of lactose results in colonies with a pinkish-purple color. Colonies of non lactose fermenters remain colorless, or at least are no darker than the color of the media.

EOSIN METHYLENE BLUE (EMB) AGAR

Pseudomonas aeruginosa Klebsiella pneumoniae

The dyes contained in this medium inhibit the growth of many accompanying Gram-positive microorganisms

LEVINE EMB BLUE AGAR

L’ agar Xylose Lysine (XL) è utilizzato per isolare i bacilli enterici Gram-negativi.

Quando l’ agar XL è

supplementato con sodio tiosolfato, citrato ammonio ferrico e sodio deossicolato viene chiamato agar XLD ^{, un}

mezzo estremamante selettivo.

La presenza di un area colorata di nero nella colonia indica la produzione di idrogeno solforato.

Salmonella sp. su XLD agar

Xylose lysine deoxycholate (XLD) agar is a highly selective and differential medium for the isolation and identification of

Additionally, Salmonella produces a positive hydrogen sulfide production reaction, which colors the centers of the colonies black

XLD

Isolation of Salmonella onto selective agar:

Sterile media (before streaking):

Plates positive for Salmonella after incubation:

Note: If the agar media change in color and the colonies are not dark (e.g., yellow), bacteria OTHER than Salmonella were isolated

Kligler Iron Agar (KIA)

& Triple Sugar Iron (TSI) Agar

Lysine Iron Agar (LIA)

Motility Indole Ornithine (MIO) Medium

source of amino acids which may be

deaminated (alkaline rx.)

peptone,

proteose peptone, beef extract, yeast extract

peptone,

yeast extract, lysine

peptone,

yeast extract, ornithine

amino acid added to note its

decarboxylation (alkaline rx.)

none lysine ornithine

fermentable sugar(s) (acid rx.)

lactose (1%),

sucrose (1% – in KIA),

glucose (0.1%) glucose (0.1%) glucose (0.1%) pH indicator phenol red:

net acid = yellow, net alkaline = red

brom-cresol purple:

net acid = yellow, net alkaline = purple

brom-cresol purple:

net acid = yellow, net alkaline = purple

source from which

H₂S may be produced sodium thiosulfate sodium

thiosulfate none indicator of H₂S

production ferrous sulfate ferric ammonium

citrate none

TRIPLE SUGAR IRON (TSI) AGAR

Triple sugar iron (TSI) agar is a medium used in the identification of Gram-negative enteric rods.

•The medium measures a bacterium's ability to utilize three sugars, glucose, sucrose and lactose.

•A pH indicator incuded in the medium can detect acid production from fermentation of these carbohydrates.

• A yellow color change indicates acid in the medium

while no color change indicates an alkaline surrounding.

TRIPLE SUGAR IRON (TSI) AGAR

Inoculation of the tube is a two step procedure. First, a loop of bacteria is spread across the surface of the agar. Second, a needle of bacteria is inserted (stabbed) into the bottom (butt) of the tube.

Carbohydrate uilization can be determined through analysis of the extent of acid production.

Acid production limited to only the butt of the tube is indicative of glucose utilization. This is because the concentration of

Acid production in the slant and butt indicates sucrose or lactose fermentation because of the relatively high concentrations of

TSI agar can also detect reduction of sodium thiosulfate to

hydrogen sulfide. Hydrogen sulfide production will turn parts of

the agar black. Production of other gases is marked by cracks in

the agar as well as an air gap at the bottom of the test tube.

INTERPRETATION OF TUBES ABOVE TUBE 1

(UNINOCULATED) TUBE 2 TUBE 3 TUBE 4 TUBE 5

SLANT - A A K K

BUTT - A A A A

HYDROGEN

SULFIDE - - - + +

GAS - - + - -

A=Acidic K=Alkaline

TRIPLE SUGAR IRON (TSI) AGAR

corresponding tube no.

above 1 2 3 4* 5

deamination of amino acids

(aerobic alkaline rx.) + + + + +

glucose fermentation

(minor acid rx.) – + + + +

lactose and/or sucrose fermentation (major acid rx.)

– – – + +

H₂S production (black

color) – – + – +**

typical examples Pseudomonas (a non-enteric)

Morganella Providencia Shigella

Citrobacter Salmonella Edwardsiella

E. coli

Enterobacter Klebsiella

Proteus

E. coli lactose+

Salmonella H2S+

* Tube 4: Much gas is often seen for this tube, evidenced by cracks in the medium. Also, methyl red-negative organisms which ferment lactose and/or sucrose may show a "reversion" toward an alkaline reaction as neutral products are formed from some of the acid. Note the slight orange to red color at the tip of the slant in tube 4A. How might such a tube look at two or more days of incubation? (Regarding the methyl red test, recall the activities of enterics in MR-VP Broth which are illustratedhere.)

** Tube 5: Enough acid can be produced to cause the black iron sulfide precipitate to break down and not be seen. In this case, the tube will look like tube 4.

TRIPLE SUGAR IRON (TSI) AGAR

Kligler Iron Agar (KIA)

corresponding tube no.

above 1 2 3 4* 5

deamination of amino acids (aerobic alkaline rx.)

+ + + + +

glucose fermentation

(minor acid rx.) – + + + +

lactose fermentation

(major acid rx.) – – – + +

H₂S production (black

color) – – + – +**

typical examples Pseudomonas (a non-enteric)

Morganella Providencia Shigella

Citrobacter Salmonella Proteus Edwardsiella

E. coli

Enterobacter Klebsiella

H₂S+Salmonella lactose+ E. coli

* Tube 4: Much gas is often seen for this tube, evidenced by cracks in the medium. Also, lactose fermenters which are methyl red-negative may show a "reversion" toward an alkaline reaction as neutral products are formed from some of the acid. This appears as shown in Tube 4A where a slight reddening of the slant occurs as the alkaline deamination reaction becomes no longer over-neutralized by acid from fermentation. How might such a tube appear after two or more days of incubation? (Regarding the methyl red test, recall the activities of enterics in MR-VP Broth, illustratedhere.)

** Tube 5: Enough acid can be produced to cause the black iron sulfide precipitate to break down and not be seen. In this case, the tube will look like no. 4.

Bile esculin agar is a medium used to identifygroup D streptococci. This

group of bacteria have the ability to grow in the presence of bile.

Group D streptococci also have the ability to hydrolyze esculin.

This hydrolysis of esculin turns the medium black and denotes a positive test. Other bacteria capable of growing in the presence of bile do not turn the medium black.

BILE ESCULIN AGAR

BILE ESCULIN AZIDE AGAR

A variation of bile esculi agar, uses sodium azide to inhibit the

growth of all other Gram-positive bacteria and Gram-negative

bacteria.

Terreni selettivi per l’isolamento di Listeria

1. Listeria Fraser Broth: brodo di arricchimento selettivo contenente esculina e cloruro di litio Supplemento: contiene

La Listeria idrolizza l’esculina ad esculetina che, reagendo con gli ioni ferrici, causa l’annerimento del brodo. La presenza del cloruro di litio e di acido nalidixico inibiscono la crescita degli enterococchi che, come Listeria, idrolizzano l’esculina.

2. Palcam Agar Base: terreno selettivo per la presenza del

indicatore:

- Esculina e ferro ferroso (L. monocytogenes idrolizza l’esculina,

producendo colonie con un alone nero)

- Mannitolo e rosso fenolo (L. monocytogenes non fermenta il mannitolo; enterococchi e stafilococchi fermentano il mannitolo, determinando un viraggio dell’indicatore di pH dal rosso al giallo).

Colonie di L. monocytogenes su Palcam

L. monocytogenes in Fraser Broth

Campy-BAP is a blood agar that is highly selective for the growth of Campylobacter species. It contains several antibiotics which inhibit the growth of Gram-positve bacteria as well as most Gram-negative organisms. After the plate is inoculated, it is placed in a microaerophilic environment such as a sealed bag containing high carbon dioxide concentrations and low oxygen concentrations.

CAMPY-BAP

The high concentrations of thiosulfate and citrate and the strong alkalinity of this medium largely inhibit the growth of Enterobacteriaceae. Any coliform bacteria, which may grow, cannot metabolize sucrose. Only a few sucrose- positive Proteus strains can grow to form yellow, vibrid-like colonies. The mixed indicator thymol blue-bromothymol blue changes its colour to yellow, when acid is formed, even in this strongly alkaline medium.

TCBS AGAR:

Colonie di V. cholerae (gialle) su TCBS

(tiosolfato-citrato-sali biliari-saccarosio)

TCBS AGAR: Vibrio Selective Agar

Colonie di vibrioni saccarosio negativi su TCBS agar

Terreni per Micobatteri

LOWENSTEIN-JENSEN (PETRAGANI)

•Tuorlo d’uovo

•Glicerolo

These substances provide fatty acids and protein required for the metabolism of mycobacteria.

•Verde malachite:

inibitore selettivo della crescita altri batteri contaminanti

Terreni per Micobatteri

Colonie di M. tubercolosis su 7-H11 Uninoculated plate

of Middlebrook 7H11 Agar

Glycerol is provided as a source of carbon and energy

Malachite green is added as a selective agent, which partially inhibits the growth of other bacteria.

Enrichment contains :

• albumin to protect the tubercle bacilli against toxic agents;

• oleic acid, a fatty acid utilized in the metabolism of the organism;

•sodium chloride to maintain osmotic equilibrium;

•catalase to destroy any toxic peroxides in the medium;

•and dextrose as an energy source.

Terreni per B.cereus

PEMBA

• Nutritional component (eg: peptone)

• Mannitol

• Sodium chloride

• Magnesium sulphate

• Disodium hydrogen phosphate

• Potassium dihydrogen phosphate

• Bromothymol blue

• Sodium pyruvate

• Agar

MEYP• Nutritional components (meat extract, peptone)

• Mannitol

• Sodium chloride

• Phenol red

• Agar

SUPPLEMENTS PEMBA and MEYP

• 5% egg yolk emulsion

• Polymixin B 100,000 IU/l

Colonies of B. cereus are crenated and approximately 5mm in diameter. They have a distinctive turquoise to peacock blue colour on PEMBA and pink on MEYP surrounded by a zone of egg yolk precipitate.

PEMBA

Sodium sulfite and fuchsin inhibit the growth of gram-positive bacteria.

E. coli and coliform bacteria metabolize lactose with the production of aldehyde and acid. The aldehyde liberates fuchsin from the fuchsin-sulfite compound, the fuchsin then colors the colonies red. In the case of E. coli, this reaction is so intense that the fuchsin crystallizes out giving the colonies a permanent greenish metallic sheen (fuchsin sheen). Lactose- negative and weakly lactose-positive E. coli do not show any fuchsin sheen.

ENDO AGAR

64

C - EC AGAR

Substrato selettivo fluorogenico e cromogenico per la determinazione simultanea di E.coli e dei coliformi nelle acque e negli alimenti

Escherichia coli alla luce normale e sotto lampada di Wood.

Si osserva la positività al test dell’indolo e l’intensa fluorescenza azzurra.

Il terreno consente la crescita selettiva degli enterobatteri e di pochi altri batteri Gram negativi, essendo i batteri Gram positivi inibiti dagli agenti selettivi presenti. Tra gli enterobatteri, i coliformi possiedono l’enzima beta-galattosidasi, idrolizzano il composto X-GAL (5-bromo-4-cloro-3 indolil - beta - D - galattopiranoside) e sviluppano colonie verde-blu. Tale reazione è resa maggiormente evidente dall’IPTG presente nel terreno. Escherichia coli, oltre ad idrolizzare il composto X-GAL, idrolizza anche il MUG (4 - metilumbelliferil - beta - D – glucuronide) e sviluppa quindi con colonie verde blu, fluorescenti alla lampada di Wood e produce indolo dal triptofano, quindi positive al test dell’indolo. Tale test è eseguibile direttamente sulla piastra con una goccia di reattivo di Kovacs che vira al rosso se la prova è positiva.

m-Aeromonas Selective Agar enhances growth of nearly all Aeromonades. The supplement Ampicillin and Vancomycin partly inhibits growth of acompanying Gram-positive and Gram-negative organisms.

Aeromonades form acid from Dextrin indicated by a color change from blue to

yellow of the pH indicator Bromothymolblue.

HAVELAAR

BTB indicator in acidic, neutral, and alkaline solutions (left to right).

Thioglycollate media is used for cultivating anaerobic, microaerophilic and aerobic microorganisms and for detecting the presence of bacteria in normally sterile materials.

Thioglycollate media, formulated with yeast extract and casitone or pancreatic digest of casein, support growth of a wide variety of fastidious microorganisms having a range of growth requirements. Sodium thioglycollate lowers the oxidation-reduction potential of the media and neutralizes the antibacterial effect of mercurial preservatives in the specimen. Resazurin and methylene blue indicate the status of oxidation or aerobiosis.

Since methylene blue is toxic to some bacteria, resazurin has replaced it as the OR indicator. Dextrose is included in some formulations because most organisms show earlier and more vigorous growth in the presence of a carbohydrate. Just prior to inoculation, the medium may be autoclaved to drive off absorbed oxygen it acquired in storage.

Thioglycollate media

Actinomiceti

CHOPPED MEAT GLUCOSE MEDIUM

Chopped meat broth is used for culturing anaerobic organisms